Chlamydomonas mutants produced using rgen rnp and method for preparing pigment using the same

ABSTRACT

A new alga having an improved ability to produce a pigment is disclosed. When the alga is used, a carotenoid-based pigment, specifically, a xanthophyll can be produced by consuming less energy, so that it is possible to efficiently produce the pigment at the industrial level. The pigment can be applied as a raw material for a food, a health functional food and a medicine, which include the pigment. Since a DNA fragment is not likely to be inserted into a target base sequence or a base sequence other than the target, it is expected that the procedure of constructing the mutant is not regulated as a GMO.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. application Ser. No. 16/050,012filed on Jul. 31, 2018, which is a Continuation-in-Part (CIP) of PCTApplication No. PCT/KR2017/004268, filed on Apr. 21, 2017, which claimspriority to Korean Patent Application No. 2016-0049439, filed Apr. 22,2016, and to Korean Patent Application No. 2017-0041761, filed Mar. 31,2017, and to Korean Patent Application No. 2017-0041762, filed Mar. 31,2017, the disclosures of which are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to an alga having a pigment producingability, a dye composition containing the alga, and a method forpreparing the pigment.

BACKGROUND ART

Macular degeneration is a disease in which degeneration occurs in themacula which is a nerve tissue located in the center of the inner retinaof the eye and causes vision impairment, and since most of thephotoreceptors are gathered in the macula and a place where an image ofan object is formed is also at the center of the macula, the maculaplays a very important role in vision. The most common cause of maculardegeneration may be an increase in age (age-related maculardegeneration), and is known to be related to family history, race, andsmoking. Because the macula is responsible for central vision, ifdegeneration occurs in the macula, a decrease in vision, centralscotoma, metamorphopsia which is a symptom in which things appeardistorted, and the like occur. Macular degeneration is largelyclassified into non-exudative (dry) and exudative (wet), and thenon-exudative macular degeneration does not significantly affect visionin most cases, except for the late stage when the atrophy of the retinaand the choroid appears, and is a step in which yellow deposits calleddrusen are seen under the retina, but in the case of the exudativemacular degeneration in which subretinal hemorrhage or subretinal fluid,pigment epithelial detachment, and the like appear, when the position ofsuch a lesion is present under the macula or immediately adjacent to themacula, a drop in vision appears from the initial stage. The exudativemacular degeneration accounts for about 10 to 20% of the total cases ofmacular degeneration, but if the exudative macular degeneration is leftas it is without being treated, vision rapidly deteriorates, so thatmany patients will be blind within two years after being diagnosed withthe exudative macular degeneration. In order to prevent maculardegeneration, it is important to find an abnormality of the macula earlythrough a periodic funduscopic examination and to make an effort so asto reduce the adjustable factors such as obesity, smoking, andhypertension. Since smoking causes damage to choroidal circulationleading to a drop in antioxidant factors in blood and causes choroidalvasoconstriction to cause low oxidative damage, a patient who is at riskof macular degeneration necessarily needs to quit smoking. Further,since a macular pigment (lutein, zeaxanthin) reduces damage caused byaging and serves to maintain a healthy retina, sufficiently ingestingthe macular pigment through vegetables and fruits or takingcommercialized vitamin supplements can help in the prevention of maculardegeneration.

The macular pigment serves to reduce age-related failing eyesight causedin the central part of the retina and prevent retinal tissue damage dueto bright light, and representative examples thereof include axanthophyll as a carotenoid-based oxycarotenoid pigment produced byoxygenation of a carotenoid. Examples of a pigment belonging toxanthophylls include lutein, zeaxanthin, or the like. It is known thatlutein acts as an antioxidant that protects the inside of the eyes thatis damaged by free oxygen radicals naturally produced in the body,reduces the growth of blood vessels that supply carcinomas to killcancer cells, and has some effects on prevention of breast cancer, coloncancer, lung cancer, ovarian cancer, and skin cancer.

Animals cannot produce xanthophylls and can obtain xanthophylls onlythrough ingestion of food, but these xanthophylls are present togetherwith chlorophylls and carotenes in the green parts such as leaves,flowers, and fruits of plants. Recently, a health functional food foreye health, including xanthophylls, and the like has attractedattention.

Existing marigold flowers are representative as a raw material forzeaxanthin and lutein, and those extracted from other higher plants hasalso been studied. In addition, zeaxanthin and lutein are also producedby genetically mutating the pigment synthesis mechanism in bacteria.Studies have also been conducted to obtain these pigments frommicroalgae. Among these conventional raw materials, marigold flowershave a disadvantage in that it takes a long time to breed floweringplants for production, and have a problem in that the production unitcost is high because the production amount is not large as compared tothe land area for production.

In order to solve these problems, the development of zeaxanthin andlutein-producing algae into which a pigment synthesis mechanism isinserted using a bacterial system for replacing a higher plant systemwas carried out, but there is a problem in that a pigment obtained frombacteria is not suitable for ultimate use as a food additive. Inaddition, since genetically modified organisms (GMOs) using a geneticinsertion technology and the like are not preferred in the domesticmarket, the GMOs act as a fatal disadvantage in the food additive markerwhere consumers' perceptions are important, and likewise as in thehigher plant system, there is a problem in that a large cost ofmaintaining a bacterial culture solution, a bioreactor, or the like maybe required.

In the case of a method of obtaining these pigments from microalgae, theconventional microalgae are a wild type which is not improved, and havea limitation in being used as optimal producing algae because thecontent of lutein is constant, but the content of zeaxanthin is very lowdepending on the amount of light.

REFERENCES OF THE RELATED ART Patent Document Korean Patent ApplicationNo. 2014-7007656 DISCLOSURE Technical Problem

An object of the present invention is to provide a method capable ofreplacing a xanthophyll used as a raw material of a conventional food ora method capable of replacing a conventional raw material productionmethod, and specifically to provide a microorganism having an excellentability to produce xanthophylls, particularly, lutein and zaexanthin, acomposition including the same, and a method for preparing xanthophyllsusing the same.

Technical Solution

In order to achieve the aforementioned object, the present inventorshave made efforts to develop algae capable of solving the insufficientproductivity of wild-type or conventionally present microalgae by usingother mutations without a genetic recombination method which may be aproblem in the food industry, and as a result, developed a mutant havinga higher yield of macular pigment than a conventional Chlamydomonasreinhardtii alga and identified an optimal method for preparing apigment using the same, thereby completing the present invention.

In this regard, the present invention provides a Chlamydomonasreinhardtii mutant having a ZEP gene mutation in which a base sequencerepresented by SEQ ID NO: 2 is inserted between a 816th base and a 817thbase in a ZEP gene sequence of a Chlamydomonas reinhardtii cw15 WTrepresented by SEQ ID NO: 1 and having an ability to producexanthophylls.

Further, the present invention provides a Chlamydomonas reinhardtiimutant having a ZEP gene mutation in which a base sequence representedby SEQ ID NO: 4 is inserted between a 816th base and a 817th base in aZEP gene sequence of a Chlamydomonas reinhardtii cw15 WT represented bySEQ ID NO: 1 and having an ability to produce xanthophylls.

In addition, the present invention provides a Chlamydomonas reinhardtiimutant having a ZEP gene mutation in which a base A is inserted betweena 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonasreinhardtii cw15 WT represented by SEQ ID NO: 1 and having an ability toproduce xanthophylls.

The three Chlamydomonas reinhardtii mutants may each have an ability toproduce xanthophylls.

The three Chlamydomonas reinhardtii mutants may each have an ability toproduce one or more pigments selected from the group consisting oflutein and zeaxanthin; and chlorophyll b, chlorophyll a, and β-carotene.

Furthermore, the present invention provides a culture of theChlamydomonas reinhardtii mutant.

Further, the present invention provides a pigment composition includingone or more selected from the group consisting of a culture of themutant, a dry material thereof, and an extract thereof.

In addition, the present invention provides a composition for oraladministration, including one or more selected from the group consistingof a culture of the mutant, a dry material thereof, and an extractthereof.

Furthermore, the present invention provides a composition for feed or afeed additive, including one or more selected from the group consistingof a culture of the mutant, a dry material thereof, and an extractthereof.

Further, the present invention provides a composition for a food or foodadditive, including one or more selected from the group consisting of aculture of the mutant, a dry material thereof, and an extract thereof.

In addition, the present invention provides a method for preparing apigment using the mutant.

Furthermore, the present invention provides a method for preparing afood or feed raw material, including: culturing the mutant.

Advantageous Effects

Through the present invention, it could be confirmed that three mutantswere constructed by using the CRISPR gene scissors technology (RGENRNPs) without any introduction of an exogenous DNA in a microalgaChlamydomonas reinhardtii to knock out a ZEP gene, and the amount ofzeaxanthin which is an industrially useful pigment was significantlyincreased when cellular characteristics of the existing wild type andthe three mutants of the present invention were compared with eachother. In particular, since a DNA fragment is not likely to be insertedinto a target base sequence or a base sequence other than the target, itis expected that the procedure of constructing the mutant is notregulated as a GMO, so that it is expected that the procedure ofconstructing the mutant can create a big economic effect in terms of anindustry which produces lutein and zeaxanthin by using microalgae.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The above and other objects, features and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the accompanying drawings, in which:

FIG. 1 illustrates a general summary on the techniques and methodsapplied in the present invention;

FIG. 2 illustrates information on a zeaxanthin epoxidase (ZEP) gene ofChlamydomonas reinhardtii cw15 wild type (SEQ ID NO: 1);

FIG. 3 is a summary of five target sequences designed for targeting theChlamydomonas reinhardtii ZEP gene (CACCAGCTGCGCGACCGAGCTGG, SEQ ID NO:12; GCCGTTGCACTTCTGAAGCAGGG, SEQ ID NO: 13; TCCGGCGAACGCACCTGGATGGG, SEQID NO: 14; TGGTGGGCGCCGACGGCATCTGG, SEQ ID NO: 15;CCATGGCTTCGCAGGCATCTCGG, SEQ ID NO: 16) [5 sgRNAs were carefullydesigned within the half of a coding sequence region of a ZEP gene whichis different from any other target sites by 3 nucleotides (nt) in theentire genome and has an out-of-frame score higher than 66 by usingCas-Designer (www.rgenome.net/cas-designer/). The ‘coding sequence (CDS)position’ refers to a relative position of an excision point in a RNAtranscript. a + direction refers to a direction which is the same as atarget sequence, that is, means that the same sequence is a sequence ofRGEN, and − refers to a direction reverse to the target sequence, thatis, a sequence having a reverse complement relationship with each other,which is a sequence bound to the target sequence. The out-of-framescore’ indicates the probability of a frame shift-inducing deletionoccurring when a cleaved double-stranded DNA is repaired by amicrohomology-mediated end joining (MMEJ) pathway. The ‘# of atarget-off site’ refers to the number of sequences mismatched throughoutthe entire genome. The linking of the remaining sgRNA sequence(gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc)(SEQ ID NO: 10) to the target sequence results in the entire sgRNA];

FIG. 4 illustrates mutations of a ZEP gene induced by DNA-free RGEN RNPs[a: RGEN-transfected cell and wild-type mutation (insertion anddeletion; indel) frequencies for each sgRNA were measured by targeteddeep sequencing. The Indel frequency was measured up to about 0.46%. b:a representative mutant DNA sequence (RGEN3) obtained from the thirdsgRNA having the highest efficiency observed from the targeted deepsequencing analysis result of a, that is, TCCGGCGAACGCACCTGGATGGG (SEQID NO: 11). Various indel patterns identified from the target sequenceby the targeted deep sequencing analysis result appearing at the 3 ntupstream of the PAM sequence. The 20-bp target sequence was underlinedand the PAM sequence was indicated in bold.]. The depicted nucleotidesequences, listed from top to bottom, are SEQ ID NOs: 17-22;

FIG. 5 illustrates the Cas9 protein sequence used in Example 1 (SEQ IDNO: 9);

FIG. 6 is a set of photographs illustrating morphologicalcharacteristics of a Chlamydomonas reinhardtii cw15 wild-type alga andChlamydomonas reinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 [a: measurement ofchlorophyll (Chl) fluorescence for several hundreds of colonies to studyZEP gene knock-out, b: a photograph of cultivation in a colony state ina solid TAP medium containing agar, c: a photograph illustrating a statewhen the concentration was adjusted to the same concentration (OD 750=1)after a Chlamydomonas reinhardtii cw15 wild-type alga and Chlamydomonasreinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 were liquid-cultured in an HSmedium];

FIG. 7 identifies variations in target DNA sequences of actual ZEP genepositions in the three ZEP mutants generated by DNA-free RGEN RNPs [a:wildtype (SEQ ID NO: 23), b: ZEP mutant 1 (ΔZ1) (SEQ ID NO: 24), c: ZEPmutant 2 (ΔZ2) (SEQ ID NO: 25), d. ZEP mutant 3 (ΔZ3) (SEQ ID NO: 26)];

FIG. 8A illustrates information on the ZEP gene of the Chlamydomonasreinhardtii mutant ΔZ1 (SEQ ID NO: 3);

FIG. 8B illustrates information on the ZEP gene of the Chlamydomonasreinhardtii mutant ΔZ2 (SEQ ID NO: 5);

FIG. 8C illustrates information on the ZEP gene of the Chlamydomonasreinhardtii mutant ΔZ3 (SEQ ID NO: 6);

FIG. 9 illustrates autotrophic culture vessels;

FIG. 10 illustrates mixotrophic culture vessels;

FIG. 11 is a set of HPLC analysis graphs illustrating pigment profilesof the Chlamydomonas reinhardtii cw15 wild-type and the Chlamydomonasreinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 [neo+lor: neoxanthin+loroxanthin,vio: violaxanthin, an: antheraxanthin, lut: lutein, zea: zeaxanthin, chla: chlorophyll a, chl b: chlorophyll b, α-car: α-carotene), and β-car:β-carotene];

FIG. 12 is a graph illustrating the growth curves (the number of cellsper volume, cells/ml) of the Chlamydomonas reinhardtii cw15 wild-typeand the Chlamydomonas reinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 over time;

FIG. 13 is a set of graphs comparing the amounts of lutein andzeaxanthin pigments produced by the Chlamydomonas reinhardtii cw15wild-type (WT) and the ZEP knock-out mutants ΔZ1, ΔZ2, and ΔZ3 over time[a: the amount (mg/L) of lutein produced over time, b: the amount (mg/L)of zeaxanthin produced over time, c: the sum (mg/L) of the amounts oflutein and zeaxanthin produced over time]; and

FIG. 14 compares the contents of zeaxanthin and lutein among higherplants known to have high contents of zeaxanthin and lutein, theChlamydomonas reinhardtii cw15 wild-type (WT), and the Chlamydomonasreinhardtii ZEP knock-out mutants ΔZ1, ΔZ2, and ΔZ3.

BEST MODE

Exemplary embodiments of the present invention will be described indetail below with reference to the accompanying drawings. While thepresent invention is shown and described in connection with exemplaryembodiments thereof, it will be apparent to those skilled in the artthat various modifications can be made without departing from the spiritand scope of the invention.

However, the present invention may be modified in various forms and mayhave various forms, so that specific examples and descriptions set forthbelow are included merely for aiding the understanding of the presentinvention, and are not intended to limit the present invention to aspecific disclosure form. It should be understood that the scope of thepresent invention includes all the modifications, equivalents, andreplacements falling within the spirit and technical scope of thepresent invention.

Hereinafter, the present disclosure will be described in more detail.

The present relates to a Chlamydomonas reinhardtii mutant.

The Chlamydomonas reinhardtii is a eukaryote distributed in variousenvironments such as fresh water and oceans as a unicellular green alga(Chlorophyta), and has a doubling time of 6 to 8 hours. Further, theChlamydomonas reinhardtii is one of the microalgae model systems mostwidely distributed and can be produced in a bioreactor.

The mutant was constructed by using RGEN RNPs which is not a generalmutation treatment, that is, the CRISPR gene scissors technology inwhich exogenous DNAs are not introduced to knock out zeaxanthinepoxidase (ZEP) genes.

The mutant is a mutant (hereinafter, referred to as ΔZ1) having a ZEPgene mutation in which a base sequence (gaaattaata agactcatta tattccggcgaacgcacctg ga) represented by SEQ ID NO: 2 is inserted between a 816thbase and a 817th base in a ZEP gene sequence of a Chlamydomonasreinhardtii cw15 WT represented by SEQ ID NO: 1. That is, the mutant isa mutant having a ZEP gene mutation represented by SEQ ID NO: 3.

Further, the mutant is a mutant (hereinafter, referred to as ΔZ2 havinga ZEP gene mutation in which a base sequence (tagctctaaa acatccaggtgcgttcgccg gactatagtg agta) represented by SEQ ID NO: 4 is insertedbetween a 816th base and a 817th base in a ZEP gene sequence of aChlamydomonas reinhardtii cw15 WT represented by SEQ ID NO: 1. That is,the mutant is a mutant having a ZEP gene mutation represented by SEQ IDNO: 5.

In addition, the mutant is a mutant (hereinafter, referred to as ΔZ3)having a ZEP gene mutation in which a base A is inserted between a 816thbase and a 817th base in a ZEP gene sequence of a Chlamydomonasreinhardtii cw15 WT represented by SEQ ID NO: 1. That is, the mutant isa mutant having a ZEP gene mutation represented by SEQ ID NO: 6.

The three Chlamydomonas reinhardtii mutants of the present inventioneach have an ability to produce a pigment, specifically, an ability toproduce xanthophylls. Specifically, the three Chlamydomonas reinhardtiimutants of the present invention may have an ability to produce luteinand zeaxanthin. More specifically, the three Chlamydomonas reinhardtiimutants of the present invention may have an ability to produce one ormore pigments selected from the group consisting of lutein andzeaxanthin; and chlorophyll b, chlorophyll a, and β-carotene.

Since the mutant has a significantly high ability to produce zeaxanthinper cell as compared to the conventional Chlamydomonas reinhardtii cw15wild-type and a content of lutein and zeaxanthin which is more than 12times higher than those of higher plants known to have a high content oflutein and zeaxanthin [see FIG. 14], there is an advantage in that themutant can be effectively used as an alga for producing xanthophyll.

In a specific exemplary embodiment, it was confirmed that the mutant ofthe present invention had a significantly increased amount of zeaxanthinproduced over time as compared to the Chlamydomonas reinhardtii cw15wild-type (FIG. 13B), so that it could be seen that the mutant of thepresent invention had mycological properties having an excellent abilityto produce xanthophylls, particularly, zeaxanthin, and it was confirmedthat the mutant of the present invention could be effectively utilizedas a source of producing a xanthophyll pigment by utilizing themycological properties.

The mutant of the present invention can survive in dim light, and may becultured under light intensity conditions specifically within a range of10 to 2,000 μmol photons/m²s. The mutant cannot photosynthesize incomplete darkness which is equal to or less than the dim lightconditions, and cell can be damaged by lighting stress under excessivelighting conditions. When the mutant of the present invention iscultured under the conditions, there is an advantage in that the mutantof the present invention has an excellent growth rate while increasingthe content of a xanthophyll in the mutant.

The mutant can be appropriately grown within typical growth environments(light intensity conditions, temperature conditions, medium, and thelike) of a Chlamydomonas reinhardtii alga. Furthermore, since the mutanthas an excellent ability to accumulate zeaxanthin even under low lightintensity (FIG. 13), the mutant can be industrially and effectively as axanthophyll pigment-producing microorganism due to the excellent abilityto produce a xanthophyll, and the density thereof in a cluster isrelatively lower than other algae even under high light intensity, sothat the mutant has an effect of having an excellent efficiency ofproducing a pigment by photosynthesis in a single cell. Specifically,the Chlamydomonas reinhardtii wild-type produces almost no zeaxanthin,but the mutant of the present invention has a content of zeaxanthin,which is higher by about 50 times or more than that of the wild-type.

The mutant can be cultured in an environment capable of culturing ageneral Chlamydomonas reinhardtii alga, and specifically, it is possibleto use a culture medium capable of culturing an alga under weak lightintensity conditions. In order to culture a specific microorganism, theculture medium contains nutritional materials required by a subject tobe cultured, that is, a microorganism to be cultured, and may be aculture medium in which a material for a special purpose is additionallyadded and mixed. The medium also refers to a culture medium or a culturesolution, and is a concept encompassing all of the natural medium, thesynthetic medium or the selective medium. The Chlamydomonas reinhardtiimutant may be cultured according to a typical culture method. Forexample, the Chlamydomonas reinhardtii mutant may be cultured in an HSmedium or a TAP medium, which is a photosynthesis medium, and a carbonsource may be added. In an exemplary embodiment, it was confirmed thatin the culture solution composition environments in Table 1 in theExamples of the present invention, the mutant of the present inventionhad an excellent ability to produce zeaxanthin.

A pH of the culture medium is not particularly limited as long as the pHis within a range enabling Chlamydomonas reinhardtii to survive and begrown, and as an example, a pH of 6 or more, specifically, Chlamydomonasreinhardtii can survive within a PH of 6 to a pH of 9, and may have anoptimal growth rate at a pH of 7.0 or more and a pH of less than 8.0.

The mutant may be constructed by treating an existing mutagen or usingthe CRISPR gene scissors technology without introducing an exogenous DNAinto a wild-type strain which is not a gene recombinant mutant throughintroduction of an exogenous gene to directly introduce RGEN RNPs into atarget sequence in a ZEP gene.

The Chlamydomonas reinhardtii mutant of the present invention canaccumulate a pigment, particularly, a xanthophyll-based pigment in thecells, and can include zeaxanthin in an even higher content among thepigments, so that the alga is cultured, and thus can be effectively usedas a raw material for a food, feed, a medicine, and the like.

In this regard, the present invention relates to a culture of theChlamydomonas reinhardtii mutant.

In the present invention, “a culture” refers to a medium in which aspecific microorganism is cultured, that is, a post-culture medium, andthe culture refers to a culture including the Chlamydomonas reinhardtiimutant. Further, the culture refers to a culture including all of theconcentrate of the culture where a post-culture medium is subjected toprocessing such as concentration and drying, or the dry material of theculture. The culture can include a byproduct thereof, the preparationthereof is not limited, and as an example, the culture may be a liquidor a solid.

In order to culture a specific microorganism, the medium containsnutritional materials required by a subject to be cultured, that is, amicroorganism to be cultured, and may be a medium in which a materialfor a special purpose is additionally added and mixed. The medium alsorefers to a culture medium or a culture solution, and is a conceptencompassing all of the natural medium, the synthetic medium or theselective medium. A pH of the medium may be more than a range in which aChlamydomonas reinhardtii mutant can be grown, and may be a pH of 6 ormore as an example, and preferably a pH of 6 to 9.

Further, the present invention relates to a composition including one ormore selected from the group consisting of the Chlamydomonas reinhardtiimutant of the present invention, a culture of the alga, a dry materialthereof, and an extract thereof.

The composition may be used for improving the health of a human and ananimal.

Since the mutant of the present invention has characteristics ofproducing a xanthophyll-based pigment including zeaxanthin and luteinand accumulating the pigment in vivo, the composition may be a pigmentcomposition or a xanthophyll pigment composition in this regard.

The pigment composition may be a composition in which zeaxanthin isincluded in an amount of 5 to 15 parts by weight based on 100 parts byweight of the total pigments included in the composition. According toan exemplary embodiment of the present invention, as a result ofmeasuring the content of zeaxanthin in the total pigments per each cellof the Chlamydomonas reinhardtii wild-type alga and the Chlamydomonasreinhardtii mutant, it could be confirmed that the Chlamydomonasreinhardtii mutant had a significantly high content of zeaxanthin in thepigment even when compared to the wild-type (FIG. 13C).

The pigment composition may be used as a raw material for a food orfeed, and may be used as a preparation for oral administration.

Accordingly, a pigment composition or xanthophyll pigment compositionincluding the composition or extract may be a composition for oraladministration, in that the pigment composition or xanthophyllcomposition included in a food, a medicine, feed, or the like may besupplied via an oral route.

The composition for oral administration may be included in a formulatedoral preparation by using a method publicly known in the art, such as apowder, a granule, a tablet, a pill, a sugar-coated tablet, a liquid, agel, a syrup, a slurry, and a suspension. For example, for the oralpreparation, a tablet or a purified material of sugar may be obtained byblending an active ingredient with a solid excipient, pulverizing thesame, adding a suitable auxiliary agent thereto, and then processing thesame into a granular mixture. Examples of a suitable excipient includesugars including lactose, dextrose, sucrose, sorbitol, mannitol,xylitol, erythritol, maltitol, and the like, starches including cornstarch, wheat starch, rice starch, potato starch, and the like,celluloses including cellulose, methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl cellulose, and the like, and fillers suchas gelatin and polyvinylpyrrolidone. In addition, a crosslinkedpolyvinylpyrrolidone, agar, alginic acid, sodium alginate, or the likemay be added as a disintegrating agent in some cases.

Furthermore, the composition can be added in order to achieve a purposeuse which is special for a food or feed, and thus may be a foodcomposition, a composition for a food additive, a feed composition or acomposition for a feed additive. When the composition is used for feedor a food, health in the body can be maintained or strengthened by axanthophyll pigment, particularly, zeaxanthin and lutein produced by theChlamydomonas reinhardtii mutant and accumulated in the cells.Specifically, the zeaxanthin and lutein can prevent or alleviatedegeneration of the macula, and the like as a macular pigment, and thusare effective for preventing or alleviating eye disorders related to themacular degeneration. More specifically, since the zeaxanthin and luteinhave effects of strengthening or maintaining eye health; preventing oralleviating the macular degeneration; preventing or alleviatingdeterioration in eye function; alleviating or preventing damage to theretina; suppressing aging; maintaining retinal health; reducing the riskof developing the macular degeneration; or preventing or alleviatingfailing eyesight, the feed or food composition may be used for the useof preventing or alleviating the symptoms or the effects.

In the present invention, “for an additive” includes all foodcompositions as long as the food composition is a constitution in whichingredients other than the main ingredient are added to a food or feed,and a specific example thereof may be an effectively active materialhaving functionality in a food or feed or a food additive defined by theMinistry of Food and Drug Safety of Republic of Korea to be added forcoloring, preservation, and the like in a processed food.

The food may be a health functional food. More specifically, the foodmay be a food functional food for eye health.

The food, the food additive, the feed or the composition for a feedadditive may further include other effective ingredients within a rangenot impairing the activity of the Chlamydomonas reinhardtii mutant ofthe present invention, a culture of the mutant, a dry material thereof,and an extract thereof. Further, it is possible to further include anadditional ingredient such as a carrier.

In the present invention, a composition for feed may be prepared in theform of fermented feed, blended feed, a pellet, silage, and the like.The fermented feed may be prepared by including the Chlamydomonasreinhardtii mutant of the present invention, a dry fungus body of themutant, a culture of the mutant, and an extract thereof, andadditionally include various microbial bacteria or enzymes. The blendedfeed may be prepared by including various types of general feed, themutant of the present invention, a dry fungus body of the mutant, aculture of the alga, and an extract thereof and mixing the mixture. Afeed in the form of a pellet may be prepared by formulating thefermented feed or blended feed with a pellet machine. The silage may beprepared by mixing silage with a Chlamydomonas reinhardtii mutant, a dryfungus body of the mutant, a culture of the mutant and/or an extractthereof, but the use of the composition of the present invention is notlimited thereto.

The composition may be mixed with a carrier and a flavoring typicallyused in the food or pharmaceutical field and may be prepared andadministered in the form of a tablet, a troche, a capsule, an elixir, asyrup, a powder, a suspension, a granule, or the like. As the carrier,it is possible to use a binder, a lubricant, a disintegrating agent, anexcipient, a solubilizing agent, a dispersing agent, a stabilizingagent, a suspending agent, and the like. As an administration method, anoral, parenteral, or application method may be used, but preferably, itis preferred that the composition is orally administered. In addition,an administration dose may be appropriately selected depending on theabsorption degree, the inactivation rate and the excretion rate of anactive ingredient in the body, and age, gender, status, and the like ofa person to be treated. A pH of the composition can be easily changeddepending on the production conditions and the like of medicine, food,and the like in which the composition is used.

The composition may include any one selected from the group consistingof a Chlamydomonas reinhardtii mutant, a culture of the mutant, a drymaterial thereof, and an extract thereof in an amount of 0.001 to 99.99wt %, preferably 0.1 to 99 wt %, based on the total weight of thecomposition, and the content of an active ingredient may beappropriately adjusted depending on the method for using the compositionand the purpose of using the composition.

The Chlamydomonas reinhardtii mutant may be included as it is or in adried form in the composition, and the culture of the alga may beincluded in a concentrated or dried form in the composition.Furthermore, the dry material refers to a dried form of the alga or theculture thereof, and may be in the form of a powder prepared bylyophilization, and the like.

Further, the extract refers to an extract obtained by extracting aproduct from the Chlamydomonas reinhardtii mutant of the presentinvention, a culture solution thereof, or a dry material thereof, andincludes an extract using a solvent, and the like, and an extractobtained by crushing the Chlamydomonas reinhardtii mutant of the presentinvention. Specifically, the extract may be an extract obtained byextracting and separating a pigment accumulated in the cells of theChlamydomonas reinhardtii mutant of the present invention by a physicalor chemical method.

The extraction procedure may be carried out by a typical method, and asan example, a target pigment may be extracted by adding an extractionsolvent to the Chlamydomonas reinhardtii mutant of the presentinvention, homogenizing the resulting mixture, and then crushing thefungus body. After the extraction, a crushed material of the alga may beremoved through centrifugation, and the extraction solvent may beremoved by a method such as distillation under reduced pressure. Inaddition, the extraction procedure may further include a typicalpurification process. The aforementioned pigment has a property of beinginsoluble in water, and thus can be more easily extracted from the algaof the present invention.

Since the Chlamydomonas reinhardtii mutant of the present invention hasan excellent ability to produce a xanthophyll, particularly zeaxanthinat low light intensity, a compound including the mutant and a byproductthereof has effects of improving body activity, maintaining bodyfunctionality, and preventing deterioration in body functionality.Specifically, since the xanthophyll pigment is known to have an effectof suppressing macular degeneration, antioxidant and anticancer effects,and the like, the composition of the present invention may be used as araw material included in a food, a health functional food, a medicine,feed, and the like for the use of maintaining body health, specifically,maintaining the body function with which the xanthophyll pigment isassociated, preventing deterioration in body function, or improving thebody function.

Furthermore, another object of the present invention is to provide amethod for preparing a pigment using the Chlamydomonas reinhardtiimutant of the present invention.

Further, still another object of the present invention is to provide amethod for preparing a food or feed raw material, including: culturingthe Chlamydomonas reinhardtii mutant of the present invention.

When the Chlamydomonas reinhardtii mutant of the present invention isused, an amount of xanthophylls accumulated in the algae to be culturedmay be increased, so that the supply of a raw material industriallyused, and the like may be efficiently carried out.

The preparation method may include culturing the Chlamydomonasreinhardtii mutant of the present invention.

In addition, the preparation method may further include: separating theChlamydomonas reinhardtii mutant of the present invention from theculture, after the culturing of the Chlamydomonas reinhardtii mutant ofthe present invention. The separated algae may be further subjected to aprocessing step including drying.

Furthermore, the preparation method may further include: extracting apigment from the Chlamydomonas reinhardtii mutant of the presentinvention, a concentrate of the culture, or a dry material of theculture.

The culturing may be carried out in a medium under a pH condition of 6.0to 8.0. Further, the culturing may be carried out under weak lightingconditions, specifically, under light intensity conditions within arange of 10 to 2,000 μmol photons/m²s. The Chlamydomonas reinhardtiimutant of the present invention has an excellent ability to produce apigment even at low light intensity, and thus may increase the contentof xanthophylls in the body, so that an excellent accumulation ofxanthophylls may be achieved without inputting energy at high lightintensity, and as a result, the Chlamydomonas reinhardtii mutant of thepresent invention may be industrially and effectively used.

The extraction may be carried out by a typical method such as a methodfor extracting a pigment from microorganisms, and examples thereofinclude an enzyme method, an ultrasonic extraction method, a mechanicalextraction method, and the like, and are not limited thereto.

The preparation method may further include, in addition to the culturingstep, a concentrating step of increasing the content of the alga afterthe culturing and a drying step of drying the alga subjected to theconcentrating step by further reducing moisture in the alga. However,the concentrating step or the drying step is not necessarily needed, andmay be generally carried out by using a concentrating and drying method,and a machine typically used in the field to which the present inventionbelongs.

The preparation method may be carried out by further including apurification step after the extracting step, and the purification stepmay be carried out by a typical purification method in the field towhich the present invention belongs.

Xanthophylls prepared through the concentrating or drying step may beused as a raw material for a food, a health functional food, a cosmetic,a medicine, or the like.

The method for preparing xanthophylls may be carried out by adoptingother methods within a range not impairing the effects of the presentinvention.

The contents on the mutant and the composition may also be appliedmutatis mutandis to the preparation method of the present invention.

Hereinafter, the present invention will be described in detail throughthe Examples. However, the following Examples are only for exemplifyingthe present invention, and the scope of the present invention is notlimited to the following Examples. The present Examples are provided tomake the disclosure of the present invention perfect and to make aperson skilled in the art to which the present invention belongsperfectly comprehend the scope of the present invention, and the presentinvention is defined only by the scope of the claims.

EXAMPLES Example 1: Construction of ZEP Gene Knock-Out Mutant

In order to target a ZEP gene of Chlamydomonas reinhardtii (phytozome:Cre02.g082550 or NCBI: AY211267.1)[phytozome.igi.doe.gov/pz/portal.html#!gene?search=1&detail=1&method=4614&searchText=transcriptid:30785220,present at a position of 1244277-1250969 in chromosome #2], for ansgRNA, five sgRNAs which allow the induction of a microhomology-drivenframeshift mutation by using Cas-Designer (www.rgenome.net) weredesigned, and were synthesized through an in vitro transcription method.FIG. 3 is a description on the target sequences of the five sgRNAsconstructed for targeting the ZEP gene. A Cas9 protein was prepared byexpressing a recombinant Cas9 protein using E. coli, and performingpurification. A Chlamydomonas reinhardtii cw15 mt-wild-type (CC-4349)used in the experiment was secured through the Chlamydomonas ResourceCenter (chlamycollection.org)[www.chlamycollection.org/product/cc-4349-cw15-mt-goodenough-330a/]. TheChlamydomonas cells were put into a 25° C. 50-ml flask containing a TAPmedium [see Table 2], and were cultured while being irradiated withlight using a fluorescence lamp at a light intensity of 70 uE and beingshaken at 90 rpm. The concentration of the cells was measured by using aspectrophotometer, and cells during a period of actively culturing to anOD₇₅₀ of approximately 0.3 to 0.5 were used. In order to make a complexof RNPs, 200 ug of the Cas9 protein (FIG. 5, SEQ ID NO: 9) was mixedwith 140 ug of the sgRNA (SEQ ID NO: 8) in nuclease-free water, andincubated at room temperature for 10 minutes. After the complex of thebound RNPs was transformed along with 50×10⁴ Chlamydomonas reinhardtiicw15 mt-wild-type (CC-4349) cells through (voltage 600 V, Capacity 50μF) electric shock in a 4 mm electroporation cuvette by using a BioradGene Pulser Xcell™ electroporation system, the complex was subjected togDNA extraction after dark incubation for 12 hours, and analyzed byperforming targeted deep sequencing, and a single colony was obtained bydiluting a part of the complex with 2,000 cells and streaking andspreading the diluted solution on a TAP agar plate. FIG. 4 is a resultidentifying the mutation of the ZEP gene, which was induced by RGEN-RNPsdue to the targeted deep sequencing. The mutation of a target gene wasidentified by separating the single colony induced by the third sgRNA(0.456%) where the transformation efficiency was at the highest level.(Panel b of FIG. 4 illustrates the data of the targeted deep sequencingand of an experiment in which when the entire cells are collected andgDNAs are extracted and analyzed after the transformation experimentusing the RNPs, all the mutations occurring in the DNA strands of atarget site for the entire cells are analyzed, and the pattern andfrequency thereof can be seen. That is, panel b of FIG. 4 illustratespatterns of the mutation actually identified at the target site throughthe targeted deep sequencing, but it is difficult to find out a big sizechange such as insertion of 42 bp or more by a principle of the targeteddeep sequencing, and there may be a difference from a single colonyactually obtained)

After the ZEP specific knock-out mutant was produced in this manner byusing DNA-free RGEN RNPs, the Chl fluorescence with respect to all thecells was measured in the petri dish, and several estimated ZEPknock-out cell lines were selected. A circular shape indicates anestimated ZEP knock-out mutant grown on a TAP agar medium under dimlight (50 μmol photons/m²s) conditions [Panel a of FIG. 4]. NPQ/4 imageswere measured by Imaging PAM (Walz). Unicellular colonies of thewild-type (WT) and ΔZEP mutant cell lines were grown on a minimum agarmedium under dim light (50 μmol photons/m²s) conditions [Panes a and bof FIG. 6]. Among the thus-identified colonies, three mutants (ΔZ1, ΔZ2,and ΔZ3) where the content of the macular pigment was increased wereselected, and a change in target DNA sequence at the actual position ofthe ZEP position was identified from the three ZEP mutants generated byRGEN RNPs through Sanger sequencing [FIG. 7].

As illustrated in FIG. 6b , it could be confirmed that the colonies ofthe Chlamydomonas reinhardtii cw15 wild-type and the mutants ΔZ1, ΔZ2,and ΔZ3 were grown in forms and sizes similar to one another on the TAPagar plate under the same light intensity conditions. Further, in thecase of the cells liquid-cultured through photosynthesis in an HSmedium, it can be confirmed that at the same concentration of the cells,the Chlamydomonas reinhardtii wild-type exhibits a striking green color,whereas the mutants ΔZ1, ΔZ2, and ΔZ3 exhibit a green color similar to agrass color tone [panel c of FIG. 6].

Among the selected mutants, the mutant Z1 was named as Chlamydomonasreinhardtii ZEP mutant 1 (ΔZ1), and the mutant was deposited at theKorean Collection for Type Cultures (KCTC), Korea Research Institute ofBioscience & Biotechnology on Mar. 22, 2017 and given Accession No. KCTC13230BP.

Example 2: Culturing of Mutant

1) Autotrophic Culture

In the case of an autotrophic culture in which the mutant was culturedin a state where an external carbon source was not supplied and by usingonly photosynthesis, the mutant was cultured in an HS medium which is aminimum medium by supplying 5% CO₂. After a medium having thecomposition as in the following Table 1 was produced, the medium wasautoclaved and prepared, and the growth was initiated by making theconcentration become 10⁶ cells/mL in the culture solution using cells inan active growth stage. A culture vessel was supplied with bubbles frombeneath using a column made of glass as in FIG. 9, and was irradiatedwith light using a fluorescence lamp at a light intensity of 200 uE fromboth sides.

TABLE 1 HS Media Ingredients Con. in culture solution (mM or μM)) Bufferand Major Ingredients (mM) NH₄Cl 9.345 MgSO₄•7H₂O 0.08 CaCl₂•2H₂O 0.07K₂HPO₄ 8.265 KH₂PO₄ 5.29 Trace Ingredients (μM) ZnSO₄•7H₂O 765 H₂BO₃ 922MnCl₂•4H₂O 511 CoCl₂•6H₂O 7 CuSO₄•5H₂O 126 (NH₄)

•4H₂O 18 FeSO₄•7H₂O 18 EDTA disodium salt 134 Others Carbon Source 5%CO₂ bubble, 80 cc/min pH in Culture Solution 7.0 Light Intensity 200 uE

indicates data missing or illegible when filed

2) Mixotrophic Culture

In the case of performing a mixotrophic culture where the mutant wascultured by supplying both photosynthesis and a carbon source, themutant was cultured by adding acetic acid to a TAP medium. After amedium having the composition as in the following Table 2 was produced,the medium was autoclaved and prepared, and the growth was initiated bymaking the concentration become 10⁶ cells/mL in the culture solutionusing cells in an active growth stage. For the culture vessels, themutant was cultured at large volumes by using a flask or bottle made ofglass as in FIG. 10, and was stirred by using a magnetic bar. The mutantwas together irradiated with light using a fluorescence lamp at a lightintensity of 70 uE.

TABLE 2 TAP Media Ingredients Con. in culture solution (mM or μM))Buffer and Major Ingredients (mM) NH₄Cl 7.5 CaCl₂•2H₂O 0.675 MgSO₄•7H₂O0.8 K₂HPO₄ 0.62 KH₂PO₄ 0.41 Trace Ingredients (μM) EDTA•2H₂O 135FeSO₄•7H₂O 18 ZnSO₄•7H₂O 75

185 MnCl₂•4H₂O 26 CuCl₂•2H₂O 6.5 Na₂MoO₄•2H₂O 5.5 CoCl₂•6H₂O 8.5 OthersCarbon Source Glacial acetic acid, 1 ml/L Tris 2.42 g/L pH in CultureSolution 7.8 Light Intensity 70 uE

indicates data missing or illegible when filed

Example 4: Pigment Analysis of Mutant and Identification of GrowthCharacteristics

1) Pigment Analysis of Mutant

After a separation into a single colony as in Example 1, the mutant wascontinuously cultured, and a pigment analysis of each colony was carriedout by using HPLC.

Specifically, the separated single colonies were cultured in a TAPmedium under 70 μmol photons/m²s conditions for 3 days, and the specificculture conditions were carried out as under the culture conditions in2) of Example 2. From the harvested alga, a pigment was extracted byusing 80% acetone, and a centrifuged supernatant was filtered again byusing a nylon filter, and then injected into an HPLC and analyzed.

Specifically, in order to separate the pigment, the total flow rate ofthe solvent was set at 1.2 mL per minute, and Tris with a pH of 8.0 andacetonitrile were each uniformly decreased from 14% and 84% to 0% fromthe 0th minute to the 15th minute, and methanol and ethyl acetate wereincreased starting from 2% to 68% and 32%, respectively, up to the 15thminute. Thereafter, this solvent ratio was maintained as it was for 3minutes (from the 15th minute to the 18th minute), and then the ratio ofeach solvent was returned to the ratio at the start for 1 minute (fromthe 18th minute to the 19th minute), and then a post-run was performedwhile maintaining the solvent ratio as it was for the remaining 6minutes. Shimadzu LC-20A Prominence manufactured by Shimadzu Company wasused as a pump, Watera Spherisorb TMS5 (DS1 4.6×250 mm, 5 μm CartridgeColumn, USA) was used as a column, and the temperature of the column wasmaintained at 40° C. Data was analyzed by using a photodiode arraydetector (SPD-M20A, Shimadzu) as a detector, and the concentrations wereobtained by using a standard curve which quantified a carotenoid andchlorophylls a and b purchased from Agern Alle, Horsholm, Denmark (DHL)as a standard from the result in which carotenoid pigments includingzeaxanthin and chlorophyll a were detected at 445 nm and 670 nm,respectively.

FIG. 11 illustrates HPLC analysis graphs illustrating a pigment profileof each alga, and FIG. 13 illustrates a set of graphs whichquantitatively analyze the contents of zeaxanthin and lutein of eachalga grown under 200 μmol photons/m²s using HPLC.

2) Identification of Growth Rate

In order to compare the cell proliferation rates and final growthamounts of the wild-type Chlamydomonas reinhardtii alga and the mutantsΔZ1, ΔZ2, and ΔZ3, the alga and the mutants were cultured under lightintensity conditions of 200 μmol photons/m²s in a state where 5% CO2bubbles were supplied in an HS medium which is a minimum photosynthesismedium [see Table 1]. The number of initially inoculated cells was 1×10⁶cells/ml, and a growth curve was drawn by measuring the number of cellsat intervals of 12 hours for 60 hours. FIG. 12 is an experimental resultof comparing the cell growth rates through photosynthesis using carbondioxide in the wild-type (WT) and the ZEP knock-out mutants. Asillustrated in FIG. 12, it was confirmed that the mutants ΔZ1, ΔZ2, andΔZ3 had a growth rate at a level similar to the wild-type as the cultureperiod elapsed.

Example 5: Identification of Pigment Production

The contents of lutein and zeaxanthin pigments of the cells werequantitatively analyzed at intervals of 12 hours by using HPLCsimultaneously while carrying out the experiment in FIG. 12. ThroughFIG. 13, the amounts of lutein and zeaxanthin produced from theChlamydomonas reinhardtii cw15 wild-type (WT) and the ZEP knock-outmutants over time were compared with one another. When the results ofthe ZEP knock-out mutants were compared to those of the wild-type, itcould be confirmed that the amounts of lutein produced from the ZEPknock-out mutants were at a level similar to that of the wild-type, butthe amounts of zeaxanthin insignificantly present in the wild-type weregreatly increased by at least 50 times or more.

The following Table 3 and FIG. 14 compare the contents of zeaxanthin andlutein from higher plants known to have high contents of zeaxanthin andlutein with those from the Chlamydomonas reinhardtii cw15 wild-type (WT)and the ZEP knock-out mutants. For the Chlamydomonas, the content(μg/100 g) was calculated by dividing the amounts of lutein andzeaxanthin pigments by the dry weight of the cells at 36 hours in FIG.12. The pigment contents (μg/100 g) of the remaining higher plants knownto have high contents of zeaxanthin and lutein were compared by citingthe USDA National Nutrient Database for Standard Reference, Release 23(2010). When the contents of lutein and zeaxanthin were compared withthose of the higher plants, the Chlamydomonas reinhardtii wild-typeexhibited a content which is higher by at least 6 times or more (thanthat of nasturtium) and the ZEP gene knock-out mutants exhibited acontent which is higher by 12 times or more (than that of nasturtium).In particular, when the content of zeaxanthin was compared with those ofthe higher plants, the ZEP gene knock-out mutants exhibited a contentwhich is higher by at least 120 times or more (than that of orangepepper).

Through FIGS. 13 and 14, high productivities and contents wereidentified, and through this, it could be confirmed that the mutants hadhigh competitiveness in the raw material market of the lutein andzeaxanthin pigment industry.

TABLE 3 Lutein + Zeaxanthin Product (μg/100 g) Chlamydomonas, cw15-, WT274,397 Chlamydomonas, ZEP mutant 1 528,353 (ΔZ1) Chlamydomonas, ZEPmutant 2 502,520 (ΔZ2) Chlamydomonas, ZEP mutant 3 544,684 (ΔZ3)Nasturtium (yellow flowers) 45,000 Kale (raw) 39,550 Kale (cooked)18,246 Dandelion leaves (raw) 13,610 Nasturtium (leaves) 13,600 Turnipgreens (raw) 12,825 Spinach (raw) 12,198 Spinach (cooked) 11,308 Swisschard (raw or cooked) 11,000 Turnip greens (cooked) 8440 Collard greens(cooked) 7694 Watercress (raw) 5767 Garden peas (raw) 2593 Romainelettuce 2312 Zucchini 2125 Brussels sprouts 1590 Pistachio nuts 1205Broccoli 1121 Carrot (cooked) 687 Maize/corn 642 Egg (hard boiled) 353Avocado (raw) 271 Carrot (raw) 256 Kiwi fruit 122

It will be apparent to those skilled in the art that variousmodifications can be made to the above-described exemplary embodimentsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention coversall such modifications provided they come within the scope of theappended claims and their equivalents.

[Information on Deposit of Microorganism]

Name of Depository Institution: Korea Research Institute of Bioscience &Biotechnology

Accession number: KCTC13230BP

Deposit date: Mar. 22, 2017

Chlamydomonas reinhardtii ZEP mutant 1 (ΔZ1) (accession number: KCTC13230BP) was deposited with Korea Research Institute of Bioscience andBiotechnology, on Mar. 22, 2017. The subject strain has been depositedunder conditions that assure that access to the strain will be availableduring the pendency of this patent application to one determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37CFR 1.14 and 35 U.S.C. 122. The deposit will be 10 available as requiredby foreign patent laws in countries wherein counterparts of the subjectapplication, or its progeny, are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

Further, the subject deposits will be stored and made available to thepublic in accord with 15 the provisions of the Budapest Treaty for theDeposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least thirty (30)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit 20 should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

1-13. (canceled)
 14. A Chlamydomonas reinhardtii mutant having a ZEPgene mutation in which a base sequence represented by SEQ ID No. 4 isinserted between a 816th base and a 817th base in a ZEP gene sequence ofa Chlamydomonas reinhardtii represented by SEQ ID No. 1 and having anability to produce a xanthophyll.
 15. The Chlamydomonas reinhardtiimutant of claim 14, wherein the Chlamydomonas reinhardtii mutant has aZEP gene mutation represented by SEQ ID No.
 5. 16. A composition fororal administration comprising one or more selected from a groupconsisting of a culture of the mutant of claim 14, a dry materialthereof, and an extract thereof.